Accurate localization of diseases in humans, especially at advanced stages, is widely accomplished by modern imaging methods such as magnetic resonance imaging (MRI) and x-ray computed tomography (CT). Additional functional information is provided by radionuclear imaging such as positron emission tomography (PET). As medical practice moves into the molecular era, optical imaging (OI) promises to complement established imaging methods by reporting molecular events without using ionizing radiation. Uniquely, OI provides high throughput screening of molecular targets from analytical assays in solution and gels through cells and small animals, and eventual translation to humans. A powerful feature of OI is a set of photophysical mechanisms for actively reporting local molecular and physiological processes within living cells and animals. OI reporting strategies provide not only functional information but specific molecular events can be quantified and diagnostic and prognostic information can be multiplexed into a single imaging procedure. In particular, the fluorescence lifetime (FLT) of an optical probe is exquisitely sensitive to the local probe environment through perturbation of excited state properties. However concerted efforts to translate the FLT method from cells and phantom studies to imaging molecular processes in whole-body animals and humans are lacking. This is particularly true for near infrared (NIR) optical imaging that allows for assessment of tissue beyond the superficial layer. To realize fully the potential of FLT approach, there is a compelling need to develop NIR molecular probes with diverse and biologically sensitive FLTs. Equally important is the development of a NIR fluorescence lifetime diffuse optical tomography (FLT-DOT) platform to combine quantitative optical property maps with simultane- ous reconstructions of yield and lifetime. To address these problems, we will (1) design, synthesize and characterize new NIR fluorescent molecular probes with FLT range between 0.2 and 10 ns;(2) optimize methods of NIR FLT molecular probes for confocal fluorescence lifetime imaging of molecular and physiological processes in cells;(3) develop high performance FLT-DOT platform;and (4) optimize and validate quantitative in vivo imaging of FLT-NIR molecular probes in mice. PUBLIC HEALTH RELEVANCE: The goals of this project are to (1) design and prepare biocompatible near infrared (NIR) fluorophores and bioconjugates with diverse fluorescence lifetime (FLT) range of 0.2 ns to 10 ns, (2) develop in vitro model for pre- dicting in vivo FLT of the molecular probes, (3) assess imaging of molecular processes by NIR confocal fluo- rescence lifetime microscopy using the NIR fluorophores, (4) and construct a highly sensitive NIR FLT diffuse optical tomography system for quantitative whole body FLT imaging.